This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Gap junctions are plasma membrane specializations found at cell-cell appositional areas. They contain tens to thousands of channels that allow the movement of molecules of 1 kDa or less between neighboring cells. Gap junction channels are built from members of the connexin (Cx) protein family, ranging in size from ~23 kDa to ~62 kDa, although the most studied connexins are Cx26 (26 kDa), Cx32 (32 kDa) and Cx43 (43 kDa). A gap junction channel consists of two connexin hexamers (hemichannels), one from each of the interacting cells, stacked back-to-back. Single particle reconstruction is being used to synthesize a more complete view of the tertiary structure of Connexin50 (Cx50) hemichannels. Cx50 gap junctions play important roles in the lens and dysfunction or mistrafficking of Cx50 mutants results in congenital cataracts. Structural studies of gap junction channels using electron or X-ray crystallographic methods have obtained atomic or near atomic resolution structures for Cx26 and a C-terminal truncated Cx43. The rigid transmembrane and extracellular domains are resolved, but the flexible cytoplasmic loop and C-terminus have not been adequately visualized either due to disorder or truncation. In connexins larger than ~40 kDa, these cytoplasmic domains, particularly the C- terminus, often contain as many amino acids as the N-terminus, transmembrane and extracellular domains combined. These cytoplasmic domains contain important kinase regulatory domains and binding sites for PDZ domain proteins (e.g. ZO-1), tubulin, debrin (an actin-binding protein) and possibly cadherins or [unreadable]-catenin. Therefore, the crystallographic structures are missing important parts of the structure. We have made advances in imaging Cx50 pannexons using vitrified cryo-EM methods and reconstruction with single particles methods.